Glioblastoma (GBM), the most frequently occurring and aggressive primary brain tumor, remains virtually incurable. Thus, there is an urgent need to develop new therapies. Genetically modified T cells expressing chimeric antigen receptors (CARs) have the potential to serve as a unique cytotoxic tool to specifically target GBM. CAR T cell therapy has been successful for hematological malignancies, but multiple challenges posed by the brain tumor environment require a multifaceted approach for CAR T cells to succeed for GBM. To study this, we have developed a single-chain variable fragment (scFv) specific for IL13R?2, a GBM-associated tumor antigen, and have generated an IL13R?2-CAR. IL13R?2-CAR T cells only recognize IL13R?2-positive glioma cells and had anti-glioma activity in preclinical xenograft and immune-competent animal models. However, tumors eventually recurred, paralleling the situation in humans. Major causes of treatment failure include (i) the inability of CAR T cells to persist within an immunosuppressive tumor environment, (ii) antigen-loss variants when a single antigen is targeted, and (iii) the inability of CAR T cells to efficiently traffic to tumor sites due to a mismatch between chemokines produced by the tumor and chemokine receptors expressed by CAR T cells. In mechanistic studies, we have demonstrated limited IL13R?2-CAR T cell persistence and the development of antigen-loss variants. In addition, we showed in xenograft models that transgenic expression of IL15 in CAR T cells enhances their persistence and anti-glioma activity. However, these xenograft studies are limited; the goal of this R01 is to perform mechanistic studies in immune-competent animal models and evaluate genetic approaches to enhance the anti-glioma activity of IL13R?2-CAR T cells. Thus, we now hypothesize that IL13R?2-CAR T cells can be further genetically engineered to optimize their anti-GBM activity by enhancing their persistence, targeting multiple tumor antigens, and improving their trafficking to tumor sites. Aim 1 investigates whether IL15-expressing CAR T cells can resist the immunosuppressive tumor environment in syngeneic GBM models. Aim 2 optimizes CAR T cells to target both IL13R?2 and EphA2, two glioma-associated antigens. Aim 3 investigates if trafficking of CAR T cells to GBMs can be improved by the transgenic expression of CCR2, a chemokine receptor that recognizes CCL2, a chemokine produced by GBMs. At the conclusion of the grant, we will have addressed three major hurdles of CAR T cell therapy for GBM. While we will use our data to justify the development of a future clinical study utilizing optimized IL13R?2-CAR T cells for patients with GBMs; our modified approach to T cell therapy should be applicable to a broad range of solid tumors.